GABA-EXCITATORY TRANSMITTER IN DEVELOPING HYPOTHALAMUS

下丘脑发育中的 GABA 兴奋性递质

• 批准号: 6187275
• 负责人: ANTHONY N VAN DEN POL
• 金额: $ 27.27万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6187275
• 关键词: age difference; brain injury; calcium flux; calcium indicator; developmental neurobiology; disease /disorder model; gamma aminobutyrate; gene expression; genetically modified animals; glutamates; green fluorescent proteins; growth cones; hypothalamus; immunocytochemistry; laboratory mouse; laboratory rat; neurogenetics; neurotrophic factors; newborn animals; synapsins; synaptogenesis; syntaxin; tissue /cell culture; transcription factor; voltage /patch clamp

DESCRIPTION (Verbatim from the Applicant's Abstract): In adult neurons, GABA acts as the primary inhibitory transmitter. In contrast, in the developing hypothalamus GABA can be excitatory by depolarizing the membrane potential, raising cytosolic calcium, and evoking action potentials. The present proposal focuses on early synapse formation in GABAergic neurons. Converging approaches utilizing fura-2 digital calcium imaging, immunocytochemistry, Northern blot mRNA analysis, and whole cell patch clamp recording with gramididin perforations address five hypotheses. Each set of experiments tests a specific hypothesis regarding GABA's early excitatory role, using both cultured hypothalamic neurons and hypothalamic slices from rats or mice. The first set of experiments addresses the hypothesis that GABA is released from axonal growth cones prior to synapse formation, and that this release is modulated by other transmitters receptors on the growing axon. We test the hypothesis that trophic factors, specifically NT-3 and BDNF, exert rapid physiological effects at the GABA developing synapse in culture and slice. This rapid action will enhance GABA release during the period when GABA is excitatory, but this enhancing effect will disappear in older neurons. The hypothesis that activity-related release of GABA will strengthen developing GABAergic synapses by a long-lasting increase in the evoked response will be tested in cultured neurons; identification of GABAergic neurons will be aided by the use of transgenic mice that express the jellyfish gene for GFP in cultured hypothalamic GABAergic cells. The hypothesis that synaptic release of GABA enhances the expression of genes coding for synaptic proteins and transcription factors in developing, but not mature, synaptic coupled neurons will be tested with Northern blot analysis. The final set of experiments pursues our earlier work showing that GABA reverts from its adult inhibitory action to an excitatory one after neuronal injury in culture. This will be extended to test the hypothesis that injury directly to the brain will result in depolarizing actions of GABA. The hypothalamus controls body temperature, the endocrine system, circadian rhythms, the autonomic nervous system, gender differentiation, energy homeostasis, and water balance, and many of the synapses involved in these functions release GABA. GABA's excitatory actions during development are not restricted to the hypothalamus, but rather are widespread throughout the brain. Thus, what we learn from our experiments on hypothalamic neurons should have general applicability to other CNS neurons.

描述(逐字摘自申请人的摘要)：在成年神经元中，GABA 作为主要的抑制性递质。相比之下，在发展中 下丘脑GABA可以通过去极化膜电位来兴奋， 升高细胞内钙离子，并诱发动作电位。目前的建议 重点研究GABA能神经元的早期突触形成。融合的方法 利用Fura-2数字钙成像、免疫细胞化学、Northern印迹 MRNA分析和全细胞膜片钳记录 穿孔解决了五个假设。每组实验都测试一个特定的 关于GABA早期兴奋作用的假说，使用培养的 大鼠或小鼠的下丘脑神经元和下丘脑切片。第一盘 的实验解决了GABA从轴突释放的假说 突触形成之前的生长锥体，这种释放是由 生长中的轴突上的其他递质受体。我们检验了这一假设 营养因子，特别是NT-3和BDNF，发挥快速的生理效应 在GABA培养和切片中发育突触。这一快速行动将 在GABA兴奋期间促进GABA释放，但这 增强效应将在较老的神经元中消失。假设 与活动相关的GABA释放将加强GABA能突触的发育 通过长期持续增加的诱发反应将在培养的 神经元；GABA能神经元的鉴定将通过使用 表达水母绿色荧光蛋白基因的转基因小鼠 下丘脑GABA能细胞。GABA突触释放的假说 增强编码突触蛋白和转录的基因的表达 正在发育但不成熟的突触耦合神经元的因素将被测试 Northern印迹分析。最后一组实验延续了我们之前的 研究表明，GABA从其成体抑制作用恢复为 培养神经元损伤后的兴奋性损伤。这将扩展到测试 直接损伤大脑将导致去极化的假说 GABA的作用。下丘脑控制体温，内分泌 系统、昼夜节律、自主神经系统、性别 分化、能量动态平衡和水平衡，以及许多 参与这些功能的突触释放GABA。GABA的兴奋作用 在发育过程中，并不局限于下丘脑，而是 广泛存在于整个大脑。因此，我们从我们的实验中学到的 下丘脑神经元应具有对其他中枢神经系统神经元的普遍适用性。